Mutations to synaptic proteins such as those in the SHANK, neuroligin, and neurexin families have been implicated in autism. SHANK and Homer scaffolding proteins normally help organize the synapse and support a number of other macromolecules, including neurotransmitter receptors.

Credit: Adapted from Feng Lab/MIT

JUNCTION DYSFUNCTION

Mutations to synaptic proteins such as those in the SHANK, neuroligin, and neurexin families have been implicated in autism. SHANK and Homer scaffolding proteins normally help organize the synapse and support a number of other macromolecules, including neurotransmitter receptors.

Credit: Adapted from Feng Lab/MIT

Forty years ago, most people believed that autism was a condition brought on by bad parenting. The thinking was that cold women—“refrigerator mothers”—who didn’t show their children enough affection caused the kids to develop speech deficits, repetitive behaviors like hand flapping, and a preference for social isolation.

Autism has come a long way since those days. It’s now known that the condition is actually a group, or spectrum, of neurodevelopmental disorders caused mainly by genetic mutation. And researchers across the globe are today zeroing in on genes potentially involved.

Hundreds of genes have been tagged so far, and some scientists are now taking the next steps to identify what they do, what brain circuits they affect, and how the proteins they encode function. In doing so, they’re even opening the door to the development of future therapeutics.

To enable these discoveries, the researchers are engineering genetic mutations into mice and then studying both the rodents’ behavior and nerve cell activity. They are also using brand-new stem cell technology to grow neurons from autistic patients’ skin cells and examine their mechanics in a petri dish.

“Even 10 years ago, I never would’ve predicted we’d be where we are right now,” says Daniel H. Geschwind, a neurologist at the University of California, Los Angeles. Around 2001, only a handful of researchers were working on autism, he says. That’s because funding for autism research was scarce then, he adds. Scientists were looking for the genes involved, but they didn’t have the right tools to find them.

When the human genome was sequenced, everything changed.

Sequence data from thousands of families are now readily available, and researchers can screen the activity of tens of thousands of genes at once. Advocacy organizations such as Autism Speaks have brought in money for research and established public databases, such as the Autism Genetic Resource Exchange. At these repositories, scientists can share and retrieve information about the genetics, behaviors, and brain activities of autistic patients, a growing population. According to the Centers for Disease Control & Prevention, one of every 110 children in the U.S. today has autism.

The genes that scientists have solidly identified as involved in autism so far have “overwhelmingly” turned out to be those that influence the development and function of neuron-neuron junctions, or synapses, says Mriganka Sur, a neuroscientist at Massachusetts Institute of Technology. At these sites, the terminal of one neuron—the presynapse—passes a signal to the terminal of another neuron—the postsynapse—via small molecules called neurotransmitters. These signaling compounds flow from the presynapse across a small cleft and activate receptor proteins embedded in the cell membrane of the postsynapse.

According to Sur, the human brain has around 1,000 trillion synapses. That sheer number, along with their intrinsic role in the brain, makes synapses hot spots for neurological dysfunction.

“If something is wrong with neuron-neuron communication at the synapse, you can imagine that you’ll have abnormal nerve circuit activity that will lead to abnormal behavior,” says Guoping Feng, a neuroscientist, also at MIT, who has made a career of learning how the synapse works.

Right now, Feng’s lab is focused on a synaptic gene strongly implicated in autism, SHANK3. “Patients with mutations in SHANK3 display obvious autistic behaviors,” he says. SHANK3 codes for a scaffolding protein located at the very tip of the postsynapse that helps anchor neurotransmitter receptors at the nerve cell surface. To do this it forms a complex with the proteins SAPAP and PSD95. The latter of these, scientists have shown, actually holds the neurotransmitter receptors in place in the neuron’s cell membrane. SHANK3 protein and other members of the SHANK family also mesh with other scaffolding proteins, such as Homer, enabling attachment of the entire complex to the neuron’s main structural foundation, the cytoskeleton.

“So you can imagine that when you mutate the SHANK3 gene,” Feng says, “you lose the integrity of the entire structural complex.” Morever, he adds, a deletion or change to this protein has the potential to disrupt so many others, particularly signaling proteins needed for neuronal communication.

By genetically engineering various mutations into the SHANK3 gene of mice, Feng and his group have recently provided further support for its link to autism (Nature, DOI: 10.1038/nature09965). Mice with the mutations socially interact far less with fellow mice in the same cage, and some of them groom themselves so repetitively that they form lesions on their faces and necks.

Another group of proteins strongly implicated in autism and connected down the line from the SHANK3 protein in the synapse are neuroligins. This family of membrane proteins is also anchored to the postsynaptic nerve cell surface via PSD95 and is thought to help organize synapses. Neuroligins bridge the synaptic cleft by connecting with proteins called neurexins on the presynaptic side.

“Without these proteins, the synapse simply doesn’t work,” says Thomas C. Südhof, a neurologist at Stanford University School of Medicine who is trying to understand how neuroligins and neurexins function. “They seem to be multifarious devices that do some housekeeping work by making sure the pre- and postsynaptic sides of neurons are connected,” he says. “They tell each other how to line up” the two sides of the synapse, and “they tell each side where to put what.”

Single amino acid substitutions, or point mutations, in neuroligin-3, in particular, have been linked to autism. Südhof and his group recently investigated the effects in mice of the R451C mutation—an arginine-to-cysteine swap at the protein’s 451st residue. Mice engineered to have the mutation are much less social than their nonmanipulated counterparts (Proc. Natl. Acad. Sci. USA, DOI: 10.1073/pnas.1111093108).

More important, the researchers found that the same mutation increases both an excitatory signal in one region of the brain and an inhibitory signal in a different region. “There are two fundamental classes of neurons in the brain: those that when they fire excite neurons down the line and those that inhibit” neuronal signaling, Südhof explains. “The balance between the signals a neuron receives determines whether it fires.”

The group’s findings, then, support one of the current hypotheses about autism—that it is a disorder caused by an imbalance in excitatory and inhibitory signals. Further testing, Südhof says, will be needed to flesh out how this works.

On the other side of the neuron-neuron junction, in the presynapse, mutations to neurexin proteins also cause abnormal brain circuitry associated with autism.

Geschwind’s group at UCLA has been studying the effects of a gene, CNTNAP2, that codes for a member of the neurexin family. Normally, this particular neurexin seems to help cluster potassium ion channels together in the presynaptic membrane.

Along with a collaborator at Weizmann Institute of Science, in Israel, Geschwind’s team examined the behavior and brain circuitry of mice engineered to be missing the CNTNAP2 gene. These mice, the researchers found, are prone to epileptic seizures—an abnormality often associated with autism—and avoid social contact with their cage mates (Cell, DOI: 10.1016/j.cell.2011.08.040). The rodents also repetitively groom themselves, emit fewer ultrasonic calls to other mice, and display out-of-sync neuronal firing in their brains.

When the team administered risperidone, a schizophrenia drug approved by the Food & Drug Administration to treat irritability in autistic children, the CNTNAP2-deficient mice lessened their repetitive grooming. “One of the neat things about having a mouse with a mutation that can cause autistic behavior,” Geschwind says, “is that it can be used for testing drugs and looking at mechanisms—things we can’t do in the human brain that well.”

Thanks to advances in stem cell technology, autism researchers also now have human-derived models, rather than just genetically altered rodents, on which to run tests. Because scientists can’t usually study humans’ neurons directly—that would require drilling into patients’ brains on a regular basis—they have learned to grow their own human-based neurons in a petri dish.

“This seems like science fiction,” says Ricardo E. Dolmetsch, a neurobiologist at Stanford’s School of Medicine. But with induced pluripotent stem cell technology, “you can now take skin or blood cells from a person and reprogram them to make stem cells.” After taking this backward step from adult to “seedling” status, the cells get molecularly coaxed to mature into neurons. If the initial skin or blood cells are taken from autistic patients, the final neurons contain the genetic makeup of those same patients—mutations and all.

Given that mouse models aren’t perfect, Dolmetsch says, this tool is powerful. A certain genetic mutation in a mouse that does something bad might not do the same bad thing to a human, he explains. The animal models are still useful, he contends, but it is clear that being able, in a dish, to poke and prod neurons derived from autistic patients is game-changing.

Dolmetsch uses the new technology to study the neuronal effects of Timothy syndrome, a disorder characterized by autistic behavior as well as heart problems such as cardiac arrhythmia. By growing nerve cells from patients with Timothy syndrome, Dolmetsch and his group determined that those afflicted with the disorder produce elevated levels of calcium ions in their neurons (Nat. Med., DOI: 10.1038/nm.2576).

The excess calcium is likely the result of a point mutation to CACNA1C, the gene responsible for Timothy syndrome. The mutated portion of the gene codes for a subunit of the calcium ion channel Cav1.2, a mediator of neuronal firing.

Dolmetsch and his team also found that their stem-cell-derived neurons produce huge amounts of the neurotransmitters dopamine and norepinephrine. Excessive production, Dolmetsch says, seems to occur because the neurons express high levels of an enzyme that catalyzes the neurotransmitters’ synthesis. Neurons treated with the drug roscovitine, a cyclin-dependent kinase inhibitor now in clinical trials for treating cancer, showed a decrease in their excess neurotransmitter production.

Skin and blood cells can be extracted from a patient, reverted into stem cells (green), and finally nudged to become nerve cells (red). Shown are cells (with blue nuclei) from a patient with Timothy syndrome.

Credit: Dolmetsch Lab/Stanford U

GROW-A-NEURON

Skin and blood cells can be extracted from a patient, reverted into stem cells (green), and finally nudged to become nerve cells (red). Shown are cells (with blue nuclei) from a patient with Timothy syndrome.

Credit: Dolmetsch Lab/Stanford U

Like Dolmetsch, a number of researchers are trying to unlock the secrets of autism by investigating the roots of other neurological syndromes in which patients exhibit autismlike symptoms. For instance, scientists at schools such as MIT and firms such as Seaside Therapeutics have made progress in understanding and developing therapeutics for Fragile X syndrome, a mental retardation disorder caused in part by synaptic protein dysfunction. According to MIT’s Sur, these single-gene disorders are an easy place to begin research, given that most cases of autism are daunting in their complexity, involving multiple mutations to multiple genes.

Sur studies Rett syndrome, a neurological disorder that, in addition to causing autistic behavior, is characterized by stunted growth and loss of speech and motor skills. It occurs almost exclusively in females. The syndrome arises from mutations to the gene MeCP2, which lives on the X chromosome and codes for a protein thought to be involved in synapse formation.

In collaboration with Rudolf Jaenisch, a biologist at MIT, Sur recently tested insulin-like growth factor 1 (IGF-1) on mice engineered with a mutation in their MeCP2 genes. The team found that administering IGF-1, a protein already approved by FDA for treating a disorder that causes short stature in children, resulted in a number of physical improvements in the mutant mice.

For instance, after injection with IGF-1, the mice had increased life spans, improved movement, and synapses with normal structure and density (Proc. Natl. Acad. USA, DOI: 10.1073/pnas.0812394106). In addition, when treated with IGF-1, the mice displayed improved levels of the synapse protein PSD95 in certain regions of their brains. These enhancements, Sur says, suggest that MeCP2 protein is, in fact, involved in synapse maturation. IGF-1 is now in clinical trials for Rett syndrome.

Benjamin D. Philpot has also had some recent success in finding a potential therapeutic for a single-gene neurological syndrome related to autism. The professor of neuroscience at the University of North Carolina, Chapel Hill, studies Angelman syndrome, a disorder characterized by speech impairment, developmental delays, epilepsy, and hyperactivity.

The gene involved in Angelman syndrome, UBE3A, is directly linked to autism. Although mutating or deleting the gene causes Angelman syndrome, duplicating it on the chromosome causes a “classic” form of autism, Philpot says. The protein that UBE3A codes for, ubiquitin-protein ligase E3A (UBE3A), is an enzyme that marks other cell proteins for degradation.

“In the gene’s absence, substrate proteins of UBE3A accumulate to an inappropriately high level,” Philpot says. In the brain, “that disrupts synaptic function.”

Gene mutations or deletions that lead to Angelman syndrome turn off expression of the maternal copy of UBE3A, the version inherited from a person’s mother. At the same time, the paternal copy lies dormant, switched off by various molecular cues.

Philpot took advantage of that fact to develop a screening method for drugs to treat Angelman syndrome. He and his group engineered mice to coexpress fluorescent protein with paternal UBE3A. Then they tested a number of drugs on mouse neurons until they got some “hits,” indicated by nerve cell fluorescence. In these cases, the drugs switched on the nonmutated paternal copy of UBE3A, restoring expression of the missing UBE3A protein (Nature, DOI: 10.1038/nature10726).

The drug that worked best was topotecan, a chemotherapeutic approved for humans. Of course, Philpot says, further testing is needed to figure out how the drug reactivates paternal UBE3A and to decide whether it can be administered at safe levels to treat Angelman syndrome.

“But what I really like about our drug discovery approach is that we’re not going downstream of the problem,” he says. “We’re trying to tackle the problem at its core.”

Some researchers, however, don’t think it is quite time to push autism drugs into clinical trials. Stanford’s Südhof worries that not enough is yet known about the molecular pathways in autism. “What I’m afraid of is that we’ll end up with an Alzheimer’s disease situation,” Südhof says. “It has been how many years since the amyloid peptide became a target? How many clinical trials? And still there’s nothing”—no therapeutics are yet available. Too many failed trials and too much money lost, he argues, can have a negative impact, potentially preventing a field from moving forward.

Still, Südhof and many other autism researchers are optimistic that scientists will eventually better understand the inner workings of the spectrum disorder. There won’t be one common molecular pathway responsible for all of autism, MIT’s Feng says, but there may be subgroups, such as the one involving the SHANK3 protein complex. In time, he says, “we’ll have an understanding of those.”

UCLA’s Geschwind also sees the future of autism in a positive light. “A decade ago, I wouldn’t have had the guts to say that we’d understand the genetic causes of up to 20% of autism cases by now—or that we’d even begin to think about bringing drugs to market for specific causes of autism,” he says. “I have enormous hope based on that progress.”

Please do not publish rubbish research that is being funded by NIH and to promote that Autism observed today has origins in genetic mutations. This is a well funded conspiracy against our children and to protect those who have caused this. It is also to blame the sickness on patient, and to make sure that it does not trace back to system that ignored the warning signals.

AUTISM OBSERVED TODAY IS AN EPIDEMIC out break. According to epidemiology the out breaks can never have genetics as origin, if such genetic was surviving for generations. Those who try to assume this as genetic based disease state of the patient are barking at wrong tree. many of today's diagnosed Autistic have no such family history. Out breaks are usually due to viruses or their synergy. Please reach out to the critiques of these jokers, who just want to fulfill their publishing quotas that involves undisputed garbage in and similar stuff out that fulfills some program manager's needs.

This will never solve and help the first generation of patients that are reaching their adulthood. We are just busy protecting insurance companies and making sure that expenses to fix these patients does not and can not hit the medicare or medicaid.

Shame on all involved. More than half of these autistic today suffer due to the legislatively mandated inoculations - knowing full well that inoculating does not mean the protection. That protection disappears within few months or few years any way; but - it subjects a few to life sentence ---- that too without having any liability to the legislative or medical community or the society at large.

Yah, we live in democratic society ---- where children do not vote, can not lobby and will never have a chance to justice or fairness! I hope our constitutional authors and experts can cherish the sacrifice that they have helped to mandate!

I completely agree with the first half of Mike Deshpande's post above - that 20% genetic causation quote has become an urban myth, based on nothing whatsoever. This is a figure that those whose research income depends on the 'autism gene myth' love to toss around, endlessly quoting each other. Dig deep for the original source of those 20% and you will find NOTHING.

While Dr Gershwin and others in the head-in-the-sand-lets-search-for-those-elusive-genes camp are busy securingmore funds for their futile searches, the number of those affected is growing by the hour.

All this reaserch will lead to nothing unless researchers from different areas start working together, and until scientists start receiving more input from clinicians and parents on the front lines.

And all of this research will come to noting useful until autism is finally recognised and treated as a systemic whole-body disease that it is.

I completely disagree with Mike Deshpande's post above - it is simply a propaganda piece for those who are entrenched in the view that autism is caused by vaccines and who refuse to accept that there is any other explanation including genetics.
As to Natasha's comment, I would take it more seriously if it actually suggested what these "different areas" are. The article quite clearly shows that behavioural scientists are working with neuroscientists and chemists as well as autism organisations, so there are already a wide range of disciplines involved

This article is amazing in its implications for all of medicine if I understood it correctly. I wanted to send it to our son whose older child is on the autism spectrum. As a grandfather I try understand this field as a result. I had a great deal of difficulty in simply emailing this news report to him. Your editorial decisions leave a lot to be desired at times. I laud this report for those who are interested but I see no point in preventing copying when it is easy to print out. I would appreciate an explanation of why I had so much trouble. Ultimately I cut it in 3 parts and I think I sent it all.

I wonder if an article on autism exists on the internet without one or more science-denying anti-vaccine loons attacking it in the comments? Leave the science to people who recognize that it is a valid method of understanding the world, please.

Ms Pepling: I finally contacted Microsoft 800-935-5700. Their tech staff fixed WORD wonderfully. I have no idea what was wrong since I had gone the usual remove reload processes some time ago. I think it is time all of us consider software as a service not something one rents or buys. I suspect for a lot of us users this arrangement would more efficient than the current one.

Our best research to date seems to indicate that the broad set of symptoms that we call autism or autism spectrum probably have environmental and genetic components, and even the genetic contribution may be in part not hereditary but due to mutations occurring in utero. More studies are also showing that the broad spectrum has been with us at least as long as our older adult population - if we look for it in a manner similar to how we look for it in the younger children we are finding it in a similar proportion of people, i.e., about 1 % of the population. Perhaps we are better at seeing or interpreting what we see these days, and perhaps environmental and / or genetic circumstances are present in many generations, and they could be the same or different. The report represents that continuing effort on the part of well-meaning people to sort out and understand these things, and it is of note that it is appearing in C&E News.

J.Feder MD
Director of Research, Interdisciplinary Council on Developmental and Learning Disorders

Thank you, Dr. Feder, for pointing out that the genetic contribution is not limited to heredity, but includes in utero mutatations well, for example. Living in New Jersey, I recently read that the autism rate for boys born in my state has reached 1 in 50. Unfortunately the article attributed this largely to better awareness and detection, and - to a lesser extent - to heredity. It semes disingenuous to ignore environmental factors when there is only one stream in the entire state that meets clean water standards, and the population has one of the highest cancer rates in the country.
We need to consider all possibilities instead of dismissing those that we find distateful.